Tumor M2-PK: A novel urine marker of bladder cancer.

Weiya Liu,Gaurav Kaushik,Ryan Didde,Jill Hamilton-Reeves,Benjamin L Woolbright,John A Taylor,Paige Martin,Jeffrey M Holzbeierlein,Karim Pirani,Eugene K Lee,Shrikant Anant,Erika Abbott,Karl X Chai

doi:10.1371/journal.pone.0218737

Abstract

PurposeBladder cancer is a “Warburg-like” tumor characterized by a reliance on aerobic glycolysis and expression of pyruvate kinase M2 (PKM2). PKM2 oscillates between an active tetramer and an inactive dimer. We aim to further characterize PKM2, in particular PKM2 dimer, as a urinary biomarker of bladder cancer and a potential target for treatment.MethodsHTB-9, HTB-5, and UM-UC3 bladder cancer cells were assessed for proliferation under differential glucose levels using the hexosaminidase assay. Western blot and Blue-native analysis was performed for protein expression of PKM2. Shikonin, an herb that is known to bind and inhibit PKM2, was utilized to determine if PKM2 has a role in glucose usage and cellular proliferation in bladder cancer cells by caspase activity assay. Institutional review board approval was obtained to collect healthy control and bladder cancer patient urine samples. The ScheBo M2-PK EDTA Plasma Test was performed on urine samples to assess urine Tumor M2-PK values.ResultsThe three bladder cancer cell lines tested all demonstrate statistically significant increases in proliferation when exposed to higher level of glucose (200mg/dL). Similarly, low doses of glucose (25mg/dL) result in reduced proliferation. Increased cell growth in higher glucose concentration correlated with up-regulation of PKM2 protein expression. Shikonin, a PKM2 inhibitor, reduced cell proliferation and switched PKM2 isoforms from the dimer to tetramer. Lastly, dimer PKM2 (Tumor-M2PK) levels were assessed in the urine samples from bladder cancer (Bca) patients and healthy controls. Tumor M2-PK significantly correlated with the presence of BCa in our subjects.ConclusionsOur studies demonstrate the potential of PKM2, specifically the dimer (Tumor-M2PK) as a target of drug therapy and as a urinary marker for bladder cancer.

Highlights

Many malignancies, including bladder cancer, have a heightened affinity for glucose and demonstrate a shift toward an aerobic glycolysis-dependent metabolism, commonly referred to as the Warburg effect [1, 2]
The three bladder cancer cell lines tested all demonstrate statistically significant increases in proliferation when exposed to higher level of glucose (200mg/dL)
Tumor M2-PK significantly correlated with the presence of BCa in our subjects

Summary

Introduction

Many malignancies, including bladder cancer, have a heightened affinity for glucose and demonstrate a shift toward an aerobic glycolysis-dependent metabolism, commonly referred to as the Warburg effect [1, 2]. Aggressive bladder cancer cell lines increase pyruvate consumption for glycolysis and increase lactate production despite the availability of sufficient oxygen, another hallmark of this phenomenon [3]. One of the principal drivers of the Warburg effect is pyruvate kinase (PKM), the rate-limiting last step in the glycolytic pathway, which catalyzes the reaction of phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP [4]. Switching from PKM2 to PKM1 subtype results in decreased lactate production, increased oxygen consumption, and reduced ability to form lung tumor in mouse xenografts [7]

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